The axon initial segment (AIS) is critical for the generation of action potentials and the maintenance of neuronal polarity. Disruption of this domain due to disease or injury can lead to nervous system dysfunction. The voltage threshold for action potential initiation is lowest at the AIS due to the high- density clusters of voltage-gated sodium channels accumulated here through interaction with the cytoskeletal adaptor protein ankyrinG (ankG). The functional organization of the AIS depends on ankG. AIS proteins are remarkably stable with half-lives that can exceed two weeks. In contrast, the half-lives of many synaptic proteins are on the order of hours. Recent work strongly suggests that the AIS is also plastic and can adapt in response to changes in activity levels. The molecular mechanisms underlying AIS protein stability and how AIS protein levels are modulated in response to changes in neuronal activity, however, remain unknown. AIS stability may depend on events that interfere with protein degradation. One way to achieve such stability could be to prevent ubiquitination of AIS proteins. The newly identified ubiquitin-related protein UBXD4 is enriched at the AIS. UBXD4 is structurally similar to ubiquitin and is thought to either block the degradation domains of its interacting partners or to tether binding partners to a deubiquitinating enzyme. The objective of this project is three-fold: To (1) determine if protein recycling at the AIS is a ubiquitin proteasome system (UPS) mediated process, (2) determine if UBXD4 is involved in the assembly of the AIS, and (3) test the hypothesis that UBXD4 contributes to AIS stability by modulating AIS protein half-life. Action potential initiation threshold at the AIS can vary between neurons and may be modulated in an activity-dependent manner. Differences in ankG turnover rates and Nav expression could drive shifts in threshold potential. To determine if activity-dependent protein recycling at the AIS is a UPS-dependent event, the activity of cultured hippocampal neurons will be pharmacologically manipulated. Cell lysates will then be immunoblotted to detect changes in AIS protein levels and to quantify the extent to which AIS proteins are ubiquitinated. Protein levels will also be assessed by immunocytochemistry. Proteasome inhibitors will be administered to cells to determine if decreased protein degradation by the UPS increases AIS protein half-life. The role of UBXD4 in AIS assembly will be tested in cultured neurons and in vivo by introducing shRNA to silence UBXD4 expression. AIS assembly will be monitored by immunostaining. UBXD4 localization at the AIS depends on ankG. Co-immunoprecipitation and pull-down experiments with His-ankG and GST-UBXD4 fusion proteins will be used to determine if UBXD4 directly interacts with ankG. Finally, AIS protein stability will be measured after the silencing UBXD4 expression and overexpressing UBXD4. AIS protein turnover rates will be assessed by immunofluorescence. The results of this project will improve our understanding of the molecular mechanisms that stabilize the AIS and contribute to its structural plasticity in response to changes in activity.